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kant0008's Content

There have been 86 items by kant0008 (Search limited from 24-January 19)



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#7375 PCR or RT-PCR?

Posted by kant0008 on 22 September 2004 - 04:29 PM in Molecular Biology

Hi,
please correct me if I misunderstand your question.
RT-PCR is used for measuring message,you are quite right. The gene shouls almost always be there, unless you are talking special cases, like the Y chromosome, or something, so normal PCR would come up. So go with RT-PCR unless you are working with special cases, or you ar eworking with transfected plasmid DNA- then normal PCR would be ok, as long as you work wuth plamid-specifi, rather than gene specific primers (that could pick up endogenous gene).
Hope that answers your question



#7374 ligation - stick 3 pieces together

Posted by kant0008 on 22 September 2004 - 04:23 PM in Molecular Biology

Unless there is somethign reaaly weird going on in your system, no , it shoudln't:)



#7373 Help: try ligation but see nothing from the gels

Posted by kant0008 on 22 September 2004 - 04:21 PM in Molecular Biology

Hi,
do you know your plasmid and insert concentrations before you start? You shoudl be able to see at least something, even if you get degradation.
Suggestions:
Don't vortex, pipetting up and down a few times should do the trick.
Don't overheat your mix. 65 should be ok for DNA, but don't leave it too long.
I do my ligations overnight always, at 4C, I don't believe them when they say 1hr is enough :huh:
I agree with labrat, have positive controls so that you know exactly what step is failing. And do check complete digestion too.
Good luck



#7368 ligation - stick 3 pieces together

Posted by kant0008 on 22 September 2004 - 04:13 PM in Molecular Biology

hi again,
as far as I understand your cloning is directional, you are using 2 enzymes ? In that case if you do a ligation control with cut plasmid only it should not religate. So then if you run that alongside your annealed product it should run differently if your 100bp fragment is ligated, as ligated plasmid should run higher (normally) thatn cut.
Hope that helps



#7366 PCR failing

Posted by kant0008 on 22 September 2004 - 04:09 PM in Molecular Biology

Hi,
seeing your PCR failed the second time round, even with the small number of samples, it sounds to me like soemthing in your reaction has gone off. If your other PCRs (if you are running any) work, and it's just this one, then your DNA is being degraded. otherwise it could be any of your reagents, dNTPs and Taq being the first ones to check.
Try running out your DNA to check integrity, or spec it for A260.280 ratio. Also change to fresh reagents.
Good luck!



#7307 3' end of forward primer mismatch

Posted by kant0008 on 20 September 2004 - 10:34 PM in Molecular Biology

Hi,
5 bases at the end of a primer sounds like a lot, I wouldn't think it would work. Depends how long it is as well. Do you know whether it worked well for your collegue?



#7306 DNA quantification

Posted by kant0008 on 20 September 2004 - 10:31 PM in Molecular Biology

Re Noel's question,
you are right, gels are only approximate. Spectrophotometry is usually good, as long as your technique is also good, ie good pipetting if you are using a dilution (I normally use 1/50) to measure concentration, clean tubes for blanking, etc. However the spec will give you total nucleotide concentration, which might be prone to error in some cases, eg. if your product is degraded, etc.
what's the experiment you need exact conc for?



#7281 ligation - stick 3 pieces together

Posted by kant0008 on 19 September 2004 - 08:34 PM in Molecular Biology

Hey postdoc,
100bp is large enough to see, all depends on the concentration of your vector on the gel. But if you get a nice bright band for the vector, then yes, you'd expect at least a very faint band for 100bp fragment



#7198 Problem with PCR on difficult template

Posted by kant0008 on 15 September 2004 - 08:10 PM in Molecular Biology

Hi,
from personal experience AmpliTag is not a very good enzyme. At least in my hands it wasn't.
If I have problems with PCR using standard Taq (I use the one from Promega), then I go to Qiagen's HotStar. It works on most difficult templates. Also they provide something called Qsolution (some buffer I guess), which also can make a difference.
If you want to try with your Taq, add a little formamide or glycerol (work similarly to DMSO) they help with amplifying GCrich templates.
Good luck



#7127 ELISA normalisation

Posted by kant0008 on 13 September 2004 - 11:12 PM in Protein and Proteomics

Hey all,
just wondering, when you do ELISA do you dilute your samples to a uniform total protein concentration and then do the assay, or is it ok to use eg. a constant volume of sample and then work out the concentration of protein of interest later, based on a Bradford?
Thanks!



#7103 About R28 cells culture

Posted by kant0008 on 12 September 2004 - 04:56 PM in Cell Biology

Hi,
how much G418 are you using? They could have some "natural" resistance, depends on what's your dose?
Also, if they have been sitting in culture side by side with transfected lines- unlikely but possible (happened in out lab) cross-contamination can occur. Check for plasmid by PCR?
Also, someone earlier had the same problem with cells that they got from someone else- maybe you don't have all the info?
Hope this helps



#7102 Freezing and thawing of cells

Posted by kant0008 on 12 September 2004 - 04:53 PM in Cell Biology

Hi,
one thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.
Good luck!



#7101 a new molecular biology lab

Posted by kant0008 on 12 September 2004 - 04:44 PM in Molecular Biology

-microscope
-cryostat

For budget purposes you can get at least some ideas from company websites, such as Promega, Roche, Invitrogen, Qiagen, etc. Otherwise Biocompare.com is not too bad, though some prices are not up to date, but it gives you a range of manufacturers for what you need



#7029 DNA quantification

Posted by kant0008 on 08 September 2004 - 08:54 PM in Molecular Biology

Hi,
do you purify your samples from the gel before sequencing? Why not spec the DNA in that case, you can use a small aliquot of diluted DNA so you don't waste your sample.
As far as markers are concerned: as long as you know what amount of marker you load and you have all the band sizes of the marker from your manufacturer, you can work out the amount of DNA in ng of each band, and by comparing the intensities work out the APPROXIMATE amount of your PCR product. For example, total base pair sum of marker SPP1 is 43787 bases (all bands added together). Then teh top band, which is 8557 bases represents 8557/43787= 0.195 of DNA. If you load 500ng of marker, then your top band will have 0.195X500= 97ng of DNA in it.
Ok, hope this helps



#7028 Western Blots

Posted by kant0008 on 08 September 2004 - 08:42 PM in Protein and Proteomics

Incubation with milk in theory should give you a cleaner western with less background noise



#6910 checking for inserts with colony PCR

Posted by kant0008 on 01 September 2004 - 03:28 PM in Molecular Biology

Hi,
are your neds compatible after digesting with RE? Otherwise you shouldn't be getting ANY self-ligated vector.
If they are compatibel- then phosphorylate, and again- no self-anneled vector



#6865 my bands spread as a line trough the gel

Posted by kant0008 on 30 August 2004 - 08:09 PM in Protein and Proteomics

Hi,
yes, make sure there's no overloading and so leakage from one well into the next. Also make sure that your spacers and combs are the same thickness- if your combs are too thin you will get gel everywhere, and might be loading your samples into the gel, embedding everywhere, rather than neatly into wells.



#6864 checking for inserts with colony PCR

Posted by kant0008 on 30 August 2004 - 03:43 PM in Molecular Biology

Hi,
I've had this problem before, with false positives from colony PCR. Do you get a weak ot strong product in your PCR? Sometimes you might have left-over unincorporated DNA in your prep, so that the PCR gives you a positive, even though your vector doesn't have inserts. Do you run a control ligation plate with vector only, no insert? Do you get colonies there? In that case you'll have to modify your cloning procedure.



#6845 Selection of clones during transfection

Posted by kant0008 on 29 August 2004 - 11:08 PM in Cell Biology

Hello again,
if you say your cells are sensitive then you can start with a low concentration of antibiotic. Theoretically for a sensitive cell line a small amount of it should still be enough to maintain a selective pressure. You can always run a control non-transfected well, to make sure you are not getting random mutants. If you have the option I'd still try a kill curve, it might actually save you time and cells in the long run.
Good luck!



#6843 Stable transformants

Posted by kant0008 on 29 August 2004 - 08:27 PM in Cell Biology

Hi,
resistant cells will continue growing and dividing in the presence of antibiotic, and you will get more and more dead cells with time in sensitive cell lines- I do my curves over 14 days. If you are trying to pick a concentration to select stable clones with- I normally go for the one that gives you dramatic cell death by about day 5, and no live cells at all by day 14.
As far as Shilpi's question goes: I'm not familiar with your particular cell line, but cell lines differ in their susceptibility to geneticin, and for some of my cells I have gone up to 1.2mg/ml. Change media as needed, as you would in normal tissue culture.



#6715 EMSA problem

Posted by kant0008 on 22 August 2004 - 10:11 PM in Protein and Proteomics

Hi,
try running your gel at 4C if you have the option to do it, your protein-DNA complex might be labile at RT.
Also if you are getting a smear (nonspecific binding?) try decreasing your protein amount, try 1ug or even less. Also with a smear sometimes it helps to add extra non-specific competitor, like polydIdC-polydIdC, try 0.2ug for every 5ug protein. If you get no bands at all, then don't add competitor at all, see what happens.
Good luck!



#6713 Selection of clones during transfection

Posted by kant0008 on 22 August 2004 - 10:04 PM in Cell Biology

Hi,
can you find out at what concentration cells will die? Construct a kill curve? It takes about 2weeks.
Otherwise 0.4-0.8mg/ml seems to be the range for most cell lines I use



#6712 If the primer is dimer,it's ok?

Posted by kant0008 on 22 August 2004 - 09:56 PM in Molecular Biology

Hi,
I guess it depends on how much of the sequence dimerises. If it's a lot then you may not have any free primer for the PCR reaction to proceed, so you won't get a product.



#6711 Importance of volume of Flascon tubes

Posted by kant0008 on 22 August 2004 - 09:52 PM in Molecular Biology

Hi,
the area that you are growing your bacteria in makes a difference to the yield, I would definitely go with the 50ml tubes. Obviously this is going to increase your amount only, not give you growth if you have none:)



#6669 EMSA problem

Posted by kant0008 on 19 August 2004 - 03:37 PM in Protein and Proteomics

Hi,
could you specify your conditions? Do you use a kit? There are a few things you could try, like changing protein amount, running at 4C and no loading buffer, making sure your probe is ds, etc. But we need to know what methods you are currently using




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